Abstract
T cell receptors (TCRαβ) are heterodimeric proteins that recognize MHC:peptide ligands. Coding sequences of TCRαβ is determined by semi-random and imprecise joining of variable, diversity, and joining gene segments. Complementarity determining region 3 (CDR3) spans the joint between these segments and typically makes the most contact with peptide during TCR recognition, largely determining specificity. A problem in identifying TCR from cells for clinical applications has been development of high-throughput methods for obtaining CDR3 sequence information from TCRαβ pairs from individual cells. We developed novel DNA origami nanostructures to simultaneously capture TCRαβ mRNA from single cells. T cells are transfected with the origami, and TCRαβ mRNA are co-captured onto bowtie-integral probes. Individual TCR sequences are then reverse transcribed and appended to origami-specific unique bowtie oligonucleotide barcodes that can be matched in downstream next-generation high-throughput sequencing. We have validated this approach using P14 TCR transgenic T cells, expressing known TCRαβ sequences. Following the synthesis of DNA origami with TCRα and TCRβ capture probes, transfection of P14 CD8+ T cells with the scaffold resulted in the specific capture and amplification of TCR sequences. Validation using Sanger sequencing confirmed capture of barcoded TCRα and TCRβ CDR3 sequences. We have developed an approach to single cell analysis of multiple mRNA species. Using a custom bowtie dual-barcoding system, we captured and re-isolated barcoded TCR sequences from individual cells. Although our focus is on the TCR repertoire, this technology is highly adaptable and can capture sequence information of any two genes of interest.